AbstractBackgroundCotyledon micrografting represents a useful tool for studying the central role of cotyledons during early plant development, especially their interplay with other plant organs with regard to long distance transport. While hypocotyl micrografting methods are well-established, cotyledon micrografting is still inefficient. By optimizing cotyledon micrografting, we aim for higher success rates and increased throughput in the model speciesArabidopsis thaliana.ResultsWe established a cut and paste cotyledon surgery procedure on a flat and solid but moist surface which improved handling of small seedlings. By applying a specific cutting and joining pattern, throughput was increased up to 40 seedlings per hour. The combination of short-day photoperiods and low light intensities for germination and long days plus high light intensities, elevated temperature and vertical plate positioning after grafting significantly increased ‘ligation’ efficiency. In particular high temperatures affected success rates favorably. Altogether, we achieved up to 92% grafting success inA. thaliana. Reconnection of vasculature was demonstrated by transport of a vasculature-specific dye across the grafting site. Phloem and xylem reconnection were completed 3–4 and 4–6 days after grafting, respectively, in a temperature-dependent manner. We observed that plants with grafted cotyledons match plants with intact cotyledons in biomass production and rosette development.ConclusionsThis cut and paste cotyledon-to-petiole micrografting protocol simplifies the handling of plant seedlings in surgery, increases the number of grafted plants per hour and greatly improves success rates forA. thalianaseedlings. The developed cotyledon micrografting method is also suitable for other plant species of comparable size.